PROJECT SUMMARY Non-Type helper 2 (Th2, also known at type 2) asthma endotypes are poorly understood despite their association with limited response to existing asthma therapeutics. This need is particularly evident for patients with severe disease, and improved understanding of the pathobiology of these endotypes is urgent. With the support of a K01 mentored-award, we have identified a network of microRNAs associated with neutrophilic asthma, a non-T2 asthma endotype. Based on the essential role played by microRNAs in gene regulation on normal tissues and disease, we are pursuing the novel hypothesis that miR-223-3p is an essential biomarker and regulator of neutrophilic airway inflammation. MiR-223-3p is associated with severe neutrophilic asthma, and our preliminary studies have found a positive correlation between its expression and Th17 inflammation in the sputum. This finding is significant given the known role of Th17 inflammation in the development and maintenance of neutrophilic asthma via IL-17 secretion. We hypothesize that miR-223-3p is involved in the regulation of the Th17 pathway and neutrophilic airway inflammation in asthma. We will utilize sputum samples in the Yale Center for Asthma and Airway Disease (YCAAD) cohort, and in vitro models, to execute the following aims: Aim 1. To determine the expression of the miR-223-3p, Th17 cytokines, and their association with neutrophilic airway inflammation. This aim will determine the association between miR-223-3p and Th17 pathway cytokines in the sputum of patients at YCAAD. Aim 2. To determine the effect of miR-223-3p in the transcriptome and secretory proteome of airway epithelial cells. This aim will determine the regulatory effects of miR-223-3p on the airway transcriptome and secretory proteome. The identification of abundant secreted proteins will be validated in patient samples collected in Aim 1. These studies will investigate how miR-223-3p contributes through neutrophilic airway inflammation in asthma via Th17 pathway regulation and how we can potentially use a hybrid biomarker, resulting from the combination of miR-223-3p expression and Th17 cytokines, to classify patients with this distinct asthma endotype. The results derived from this project will lay the foundation for the improved identification of patients with severe neutrophilic asthma and lead to a better understanding of how miR-223-3p regulates neutrophilic airway inflammation.